Comparative evaluation of DNA extraction methods from smut fungi teliospores

Smut fungi, particularly those within the genera Tilletia, Ustilago, Sporisorium, and Urocystis, threaten cerealcrops worldwide. Accurate molecular identification of these pathogens requires high-quality genomic DNA,however, extracting DNA from smut teliospores remains a challenge due to their thick and resilient cell walls. To address this, four commonly used DNA extraction methods — Murray & Thompson, Raeder & Broda, Chelex 100
and HotSHOT — were comparatively evaluated across ten species of smut fungi: Tilletia laevis, T. caries, T.controversa, T. indica, Ustilago tritici, U. nuda, U. hordei, Sporisorium maydis, S. ehrenbergii, and Urocystis agropyri. Mechanical disruption using sterilized carborundum, with or without liquid nitrogen, was applied uniformly across all methods. The quality and quantity of extracted DNA were assessed by Nanodrop spectrophotometry and PCR amplification using ITS1/ITS4 primers. All methods yielded PCR-amplifiable DNA;however, substantial differences were observed in yield and purity. Raeder & Broda method yielded the highest average DNA concentration (1050.55 ng/μl), followed by HotSHOT (951.33 ng/μl), Murray & Thompson (918.82 ng/μl), and Chelex (879.37 ng/μl). While HotSHOT offered a higher yield than Murray & Thompson and Chelex, its lower purity suggested co-purification of cellular contaminants. Murray & Thompson and Raeder and
Broda-extracted DNA exhibited better overall purity based on their average A260/A280 and A260/A230 ratios. Electrophoresis of PCR products revealed strong and consistent bands for all samples, indicating successful DNA amplification. This study demonstrates that Murray & Thompson remains optimal when purity is crucial, while Raeder & Broda is effective for higher-yield applications. HotSHOT and Chelex, despite lower purity, remain useful for rapid and routine diagnostics.